The present invention relates generally to cytolytic T lymphocyte clones derived from colorectal cancers and methods of use thereof in therapy, assays and drug screening.
Immunotherapy has been proposed as an alternative and/or complementary approach to conventional chemotherapy and radiation therapy in the treatment of certain cancers. In light of the important role that cellular immunity may play in the control of cancer [A. Houghton, The Lancet, 345:1384-1385 (1995)], approaches to the specific modulation of patients"" T-cell responses need to be developed.
Perhaps the most promising immunotherapy involves adoptive transfer of cytolytic T lymphocytes (CTLs) and/or active immunization with CTL-defined antigens. Peptides or cloned antigens recognized by patients"" lymphocytes may provide effective cancer vaccines. For example, cytolytic T lymphocytes (CTL) induced in melanoma patients"" lymphocytes by stimulation with autologous tumor cells provided the basis for the development of antigen- and peptide-derived vaccines against this tumor type [Y. Kawakami et al, in Biologic Therapy of Cancer, 2nd edition, pp. 53-64, eds. V. T. DeVita, S. Heliman, and S. Rosenberg, Philadelphia: J. Lippincott (1995) (Kawakami III)]. Kawakami, Y., et al., J. Immunol., 154: 3961-3968 (1995) (Kawakami II) demonstrated that adoptive transfer of CD8+ CTL against melanoma-associated antigen gp 100 induced tumor regression in the autologous patient. The CTLs had been induced by stimulating the patient""s tumor-infiltrating lymphocytes with autologous, fresh tumor cells.
Descriptions of known CD4+ CTL which are able to lyse autologous cells in a human leukocyte antigen (HLA) class I dependent manner [Hayashi, Y., et al., Cell Immunol., 139 411-425 (1992); L. LeMay et al, Cancer Immunol. Immunother., 37:187-194 (1993); D. Kharkevitch et al, Int. J. Cancer, 58:317-323 (1994); T. Morisaki et al, Cancer Immunol. Immunother., 39:172-178 (1994)], have reported that target cell lysis by the CD4+ CTL was slow (requiring overnight incubation of CTL and targets).
Colorectal carcinoma (CRC) is, after lung carcinoma, the most common type of cancer in the United States. However, attempts to establish CTL against colorectal carcinoma (CRC) by stimulating CRC patients"" lymphocytes with autologous tumor cells have been unsuccessful so far. The T cells induced in CRC patients by stimulation of the patients"" lymphocytes with autologous tumors have usually been non-cytolytic [B. Patel et al, Int. J. Cancer, 51:878-885 (1992); S. Horn et al, Cancer Immunol. Immunother., 36:1-8 (1993); J. Ransom et al, Int. J. Cancer, 54:734-740 (1993); S. Somers et al, Surg. Oncol., 2:283-291 (1993); and W. Mulder et al, Cancer Immunol. Immunother., 41:293-301 (1995)].
In two studies, CTL induction by specific peptide stimulation in CRC patients"" lymphocytes was demonstrated. In one study, induction of CTL against CRC required specific in vitro stimulation of the lymphocytes with a defined peptide, mutated p21 ras [B. Fossum et al, Int. J. Cancer, 56:40-45 (1994) (Fossum I) and B. Fossum et al, Cancer Immunol. Immunother., 40:165-172 (1995) (Fossum II)]. Furthermore, the resulting CTL induced against mutated p21 ras lysed only interferon (IFN)-xcex3-treated (but not untreated) allogeneic CRC cells, and did not lyse autologous tumor cells.
In the second study, induction of CTL against CRC required specific in vivo and in vitro stimulation of the lymphocytes with carcinoembryonic antigen [K. Tsang et al, J. Natl. Cancer Inst., 87:982-990 (1995)]. The lytic activity of the resulting CTL directed against carcinoembryonic antigen was low, requiring prolonged in vitro stimulation of the CTL and long-term (12-18 hours) incubation to demonstrate lysis by a 51Cr-release assay.
Both proliferative and cytolytic functions of T lymphocytes directed against tumor cells of various tissue origins have been previously shown to decrease with progression of the disease. Primary, but not metastatic, tumors stimulate patients"" autologous T cells in culture [D. Guerry et al, J. Clin. Invest., 73:267-271 (1984) (Guerry I) and D. Guerry et al, J. Immunol., 139:305-312 (1987) (Guerry II)]. T cells from animals and patients with less advanced tumors can lyse autologous tumor cells in vitro [J. Travis, Science 258:1732-1733 (1992) and C. Loeffler et al, J. Immunol., 149:949-956 (1992)).
Defective lymphokine production and decreased expression of the xcex6 chain by lymphocytes infiltrating metastatic tumors have been suggested to underlie the in vivo failure of these lymphocytes to control tumor development [H. Mizoguchi et al, Science, 258:1795-1798 (1992); J. Finke et al, Cancer Res., 53:5613-5616 (1993); H. Nakagomi et al, Cancer Res., 53:5610-5612 (1993); and S. Salvadori et al, J. Immunol., 153:5176-5182 (1994)]. T cells derived from peripheral blood mononuclear cells (PBMC) showed higher expression of xcex6 chain as compared to the tumor-infiltrating T lymphocytes derived from the same patient [Finke, J. H., et al., Cancer Res., 53: 5613-5616 (1993) and H. Nakagomi et al, cited above]. Moreover, advanced (metastatic) tumors, including colon carcinomas, often show reduced expression of MHC class I and class II antigens [C. Cordon-Cardo et al, Cancer Res., 51:6372-6380 (1991); A. Csiba et al, Br. J. Cancer, 50:699-709 (1984); and F. Momburg et al, Int. J. Cancer, 37:179-184 (1986)].
There exists a need in the art for reagents and methods useful for successful immunotherapy regimens for the treatment of colorectal cancers.
In one aspect, the invention provides a stable cytolytic T lymphocyte (CTL) cell line established from the peripheral blood mononuclear cells (PBMC) of a patient with minimal residual colorectal carcinoma (CRC) following removal of the primary lesion. The CTL cell line not only lyses the autologous primary CRC, but also lyses allogeneic, HLA-matched metastatic CRC. One such cell line is referred to as CTL007. A therapeutic reagent containing such a cell line in a suitable pharmaceutical carrier is also provided.
In another aspect, the invention provides a method for generating a stable colorectal carcinoma CTL clone by stimulating the lymphocytes of a patient with minimal or no clinical evidence of colorectal carcinoma following removal of the primary lesion in culture with irradiated autologous primary CRC tumor cells and interleukin-2 in the presence of feeder cells, which may be autologous peripheral blood lymphocytes (PBLs) or a lymphoblastoid cell line (LCL), such as Epstein Barr virus transformed B (EBV-D) cells.
In another aspect, the invention provides a therapeutic or diagnostic product derived or produced from, or utilizing, a CTL cell line of this invention. For example, in one embodiment, the invention provides a CRC-associated CTL antigen or peptide fragment identified by the CTL cell line described above, and an immunotherapeutic composition useful in the treatment of CRC containing the antigen or peptide fragment. In another embodiment, the invention provides a factor which is produced by, or secreted by, the cell line. Still other products may be derived from use of the cell lines of this invention.
In another aspect, the invention provides for the identification of a tumor-associated antigen or peptide thereof with high potential for inducing CTL responses in a patient by using a CTL cell line of the invention in a conventional assay known to one of skill in the art.
In yet a further aspect, the invention provides a method for treating a patient with CRC comprising administering to said patient an effective amount of a CTL cell described above.
In still another aspect, the invention provides a method for inducing CTL by stimulating a CRC patient""s lymphocytes with autologous CRC tumor cells and IL-2.
In still another aspect, the invention provides a method for bone marrow purging and adoptive transfer therapy of CRC in a patient, by treating bone marrow cells of the patient with a suitable amount of a CTL cell as described herein and reinjecting the treated bone marrow cells into the immunosuppressed patient.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof